Crosslinkable biomaterial compositions containing hydrophobic and hydrophilic crosslinking agents

ABSTRACT

The present invention discloses novel crosslinked biomaterial compsotions which are prepared using hydrophobic polymers as a crosslinking agent. Preferred hydrophobic polymers are those that contain two or more reactive succinimidyl groups, including disuccinimidyl suberate, bix(sulfosuccinimidyl) suberate, and dithiobis(succinimidylpropionate). Crosslinked biomaterial compositions prepared using mixtures of hydrophobic and hydrophilic crosslinking agents are also disclosed. The compositions of the present invention can be used to prepare formed implants for use in a variety of medical applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/987,467, filed Dec. 9, 1997, abandoned, which was a continuation ofU.S. application Ser. No. 08/403,358, filed Mar. 14, 1995, alsoabandoned, the disclosures of which areincorporated-herein-by-reference.

FIELD OF THE INVENTION

This invention relates generally to the use of hydrophobic crosslinkingagents to prepare injectable or implantable crosslinked biomaterialcompositions for use in a variety of therapeutic applications.Specifically, this invention relates to crosslinked biomaterialcompositions prepared using hydrophobic crosslinking agents containingtwo or more succinimidyl groups, such as disuccinimidyl suberate,bis(sulfosuccinimidyl) suberate, or dithiobis(succinimidylpropionate).Also provided are unique crosslinked biomaterial compositions preparedusing-mixtures of hydrophobic and hydrophilic crosslinking agents. Thecompositions of the invention are particularly useful in the preparationof formed implants for a variety of medical uses.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,162,430, issued Nov. 10, 1992 to Rhee et al., andcommonly owned by the assignee of the present application, disclosescollagen-synthetic polymer conjugates and methods of covalently bindingcollagen to synthetic hydrophilic polymers. Commonly owned U.S. Pat. No.5,328,955, issued Jul. 12, 1994 to Rhee et al., discloses variousactivated forms of polyethylene glycol and various linkages which can beused to produce collagen-synthetic polymer conjugates having a range ofphysical and chemical properties. Commonly owned U.S. Pat. No.5,324,775, issued June 28, 1994 to Rhee et al., discloses biocompatiblepolymer conjugates prepared by covalently binding biologically inert,biocompatible polymers to synthetic hydrophilic polymers.

Commonly owned, copending U.S. application Ser. No. 08/146,843, filedNov. 3, 1993, discloses conjugates comprising various species ofglycosaminoglycan covalently bound to synthetic hydrophilic polymers,which are optionally bound to collagen as well. Commonly owned,copending U.S. application Ser. No. 08/147,227, filed Nov. 3, 1993,discloses collagen-polymer conjugates comprising chemically modifiedcollagens such as, for example, succinylated collagen or methylatedcollagen, covalently bound to synthetic hydrophilic polymers to produceoptically clear materials for use in ophthalmic or other medicalapplications.

Hydrophobic crosslinking agents such as such as disuccinimidyl suberate,bis(sulfosuccinimidyl) suberate, and dithiobis(succinimidylpropionate)have a long history of use for crosslinking biologically activepeptides, as described in the 1992 Pierce (Rockford, Ill.) catalog.

All publications cited above and herein are incorporated herein byreference to describe and disclose the subject matter for which it iscited.

We now disclose a detailed description of preferred embodiments of thepresent invention, including crosslinked biomaterial compositionsprepared using various hydrophobic crosslinking agents and crosslinkedbiomaterial compositions prepared using mixtures of hydrophobic andhydrophilic crosslinking agents.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structural formula for disuccinimidyl suberate (DSS),and a reaction product obtained by reacting DSS with collagen.

FIG. 2 shows the structural formula for dithiobis(succinimidylpropionate) (DSP), and a reaction product obtained by reacting DSP withcollagen.

FIG. 3 shows the structural formula for bis(sulfosuccinimidyl)suberate(BS³), and a reaction product obtained by reacting BS³ with collagen.

FIG. 4 shows the structural formula forbis(2-succinimidooxycarbonyl-oxy)ethyl sulfone (BSOCOES), and a reactionproduct obtained by reacting BSOCOES with collagen.

FIG. 5 shows the structural formulas for3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP), and a reactionproduct obtained by reacting DTSSP with collagen.

FIG. 6 shows a reaction scheme for derivatizing trimethylpropane to itstricarboxylic acid form, then further derivatizing this tricarboxylicacid from by reacting it with N-hydroxysuccinimide (NHS) in the presenceof N,N′dicyclohexylcarbodiimide (D CC) to produce a trifunctionalcrosslinking agent.

FIG. 7 shows a reaction scheme for derivatizing di(trimethylpropane) toits tetracarboxylic acid form, then further derivatizing thistetracarboxylic acid form by reacting it with NHS in the presence of DCCto produce a tetrafunctional crosslinking agent;

FIG. 8 is a bar graph illustrating the wet weight recovery of implantsof various compositions at day 7 (solid-bars), day 14 (hatched bars,heavy lines), day 28 (stippled bars) and day 90 (hatched bars, lightlines).

FIG. 9 depicts the method used to measure the mechanical force requiredto dislodge implants of various compositions from the surroundingtissues. The arrows indicate the direction of the applied force.

FIG. 10 is a graph showing the anchoring force, in newtons, of implantsof various compositions removed at days 7, 14, 28 and 90.

SUMMARY OF THE INVENTION

In our earlier patents and applications, we disclosed variouscrosslinked biomaterial compositions prepared using synthetichydrophilic polymers, preferably functionally activated polyethyleneglycols (PEGs), as the crosslinking agent. In accordance with thepresent invention, we have since discovered that various hydrophobicpolymers containing two or more succinimidyl groups, such asdisuccinimidyl suberate, bis(sulfosuccinimidyl) suberate, ordithiobis(succinimidyl-propionate), can be used to crosslink variousbiomaterials such as collagen and glycosaminoglycans. We have alsodiscovered that certain hydrophobic polymers, such as polyacids, can bederivatized to contain two or more succinimidyl groups and, in thederivatized form, can be used to crosslink collagen andglycosaminoglycans. Furthermore, we have discovered that uniquecrosslinked biomaterial compositions can be prepared by using a mixtureof hydrophobic and hydrophilic crosslinking agents.

The present invention pertains to conjugates comprising biomaterialscovalently bonded to hydrophobic polymers, wherein the hydrophobicpolymer contains two or more succinimidyl groups prior to bonding withthe biomaterial. Included in the invention are conjugates comprisingbiomaterials covalently bonded to hydrophobic polymers, in which thehydrophobic polymer has been chemically derivatized to contain two ormore succinimidyl groups prior to bonding with the biomaterial.Heterogeneous crosslinked biomaterial compositions are also disclosedwhich comprise a biomaterial (or mixtures of different species ofbiomaterials), a hydrophobic crosslinking agent, and a hydrophiliccrosslinking agent. Further, in accordance with the invention, formedimplants are prepared using the conjugates and compositions of theinvention.

The compositions of the present invention have many unique andunexpected features when compared with the previously disclosedcrosslinked biomaterial compositions prepared using only hydrophiliccrosslinking agents. An important feature of the compositions of thepresent invention (when compared to previous crosslinked biomaterialcompositions) is slower degradation, resulting in greater chemicalstability, which may lead to increased in vivo persistence. Additionalfeatures and advantages of the invention will become apparent uponreading the detailed description of the invention which follows.

Definitions

It must be noted that, as used in this specification and the appendedclaim, the singular forms “a”, “an”, and “the” include plural referentsunless the context clearly dictates otherwise. For example, reference to“a conjugate” includes one or more conjugate molecules, reference to “anarticle” includes one or more different types of articles known to thoseskilled in the art and reference to “the collagen” includes mixtures ofdifferent types of collagens and so forth.

Specific terminology of particular importance to the description of thepresent invention is defined below:

The term “atelopeptide collagen” refers to collagens which have beenchemically treated or otherwise processed to remove the telopeptideregions, which are known to be responsible for causing an immuneresponse in humans to collagens from other animal, such as bovine,sources.

The term “biomaterial” as used herein refers in general tobiocompatible, naturally occurring polymers, including collagen,gelatin, and polysaccharides such as glycosaminoglycans.

The terms “chemically conjugated” and “conjugated” as used herein meanattached through a covalent chemical bond. In the practice of theinvention, a hydrophobic polymer and a biomaterial may be covalentlyconjugated to each other by means of a reactive succinimidyl group onthe hydrophobic polymer.

The term “chemical crosslinking agent” as used herein refers to anychemical agent capable of covalently binding biomaterials, such ascollagen, glycosaminoglycans, and mixtures thereof, to form acrosslinked biomaterial network.

The term “collagen” as used herein refers to all types and forms ofcollagen, including those which have been recombinantly produced,extracted from naturally occurring sources (such as bovine corium orhuman placenta), processed, or otherwise modified.

The term “collagen suspension” refers to a suspension of noncrosslinkedor crosslinked collagen fibers in an aqueous carrier, such as water orphosphate-buffered saline (PBS) solution.

The term “collagen-synthetic polymer” refers to collagen covalentlybonded to a synthetic hydrophilic polymer. For example, “PEG-collagen”denotes a composition of the invention wherein molecules of collagen arecovalently bonded to molecules of polyethylene glycol (PEG).

The term “difunctionally activated” refers to synthetic hydrophilicpolymer molecules which have been chemically derivatized so as to havetwo functional groups capable of reacting with primary amino groups onbiocompatible polymer molecules, such as collagen or deacetylatedglycosaminoglycans. The two functional groups on a difunctionallyactivated synthetic hydrophilic polymer are generally located atopposite ends of the polymer chain. Each functionally activated group ona difunctionally activated synthetic hydrophilic polymer molecule iscapable of covalently binding with a biocompatible polymer molecule,thereby effecting crosslinking between the biocompatible polymermolecules.

The term “dry” means that substantially all unbound water has beenremoved from a material.

The term “fibrillar collagen” refers to collagens in which the triplehelical molecules aggregate to form thick fibers due to intermolecularcharge and hydrophobic interactions.

The term “functionally activated” refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have one ormore functional group capable of reacting with primary amino groups onbiocompatible polymer molecules.

The term “in situ” as used herein means at the site of administration.

The term “in situ crosslinking” as used herein refers to crosslinking ofa biocompatible polymer implant following implantation to a tissue siteon a human or animal subject, wherein at least one functional group onthe synthetic polymer is covalently conjugated to a biocompatiblepolymer molecule in the implant, and at least one functional group onthe synthetic polymer is free to covalently bind with, otherbiocompatible polymer molecules within the implant, or with collagenmolecules within the patient's own tissue.

The term “molecular weight” as used herein refers to the weight averagemolecular weight of a number of molecules in any given sample, ascommonly used in the art. Thus, a sample of PEG 2000 might contain astatistical mixture of polymer molecules ranging in weight from, forexample, 1500 to 2500, with one molecule differing slightly from thenext over a range. Specification of a range of molecular weightindicates that the average molecular weight may be any value between thelimits specified, and may include molecules outside those limits. Thus,a molecular weight range of about 800 to about 20,000 indicates anaverage molecular weight of at least about 800, ranging up to about20,000.

The term “multifunctionally activated” refers to synthetic hydrophilicpolymers which have been chemically derivatized so as to have two ormore functional groups which are located at various sites along thepolymer chain and are capable of reacting with primary amino groups onbiocompatible polymer molecules. Each functional group on amultifunctionally activated synthetic hydrophilic polymer molecule iscapable of covalently binding with a biocompatible polymer molecule,thereby effecting crosslinking between the biocompatible polymermolecules. Types of multifunctionally activated hydrophilic syntheticpolymers include difunctionally activated, tetrafunctionally activated,and star-branched polymers.

The term “noncrosslinked collagen” refers to collagens that have notbeen previously crosslinked using chemical crosslinking agents. Suchnoncrosslinked collagens may include both fibrillar and nonfibrillarcollagens.

The term “nonfibrillar collagen” refers to collagens in which the triplehelical molecules do not aggregate to form thick fibers, such that acomposition containing nonfibrillar collagen will be optically clear.

The terms “synthetic hydrophilic polymer” or “synthetic polymer” referto polymers which have been synthetically produced and which arehydrophilic, but not necessarily water-soluble. Examples of synthetichydrophilic polymers which can be used in the practice of the presentinvention are polyethylene glycol (PEG), polyoxyethylene, polymethyleneglycol, poly-trimethylene glycols, polyvinylpyrrolidones,polyoxyethylene-polyoxypropylene block polymers and copolymers, andderivatives thereof. Naturally occurring polymers such as proteins,starch, cellulose, heparin, hyaluronic acid, and derivatives thereof areexpressly excluded from the scope of this definition.

The term “tissue augmentation” as used herein refers to the replacementor repair of defects in the soft or hard tissues of a human body.

Except as otherwise defined above, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Although anymethods and materials similar or equivalent to those described hereinmay be useful in the practice or testing of the present invention, onlythe preferred methods and materials are described below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In accordance with the present invention, unique crosslinked biomaterialcompositions are prepared using hydrophobic crosslinking agents, ormixtures of hydrophilic and hydrophobic crosslinking agents. In order toprepare the crosslinked biomaterial compositions of the presentinvention, it is first necessary to provide one or more biomaterials anda hydrophobic crosslinking agent.

Preferred Biomaterials

Any biomaterial that has, or can be chemically derivatized to have,primary amino (—NH₂) groups capable of binding with hydrophobic orhydrophilic crosslinking agents according to the methods of theinvention may be used to prepare the crosslinked biomaterialcompositions of the invention. Preferred biomaterials for use in thepractice of the present invention include all types of collagen andglycosaminoglycans, and mixtures thereof.

In general, collagen from any source may be used in the practice of thepresent invention; for example, collagen may be extracted and purifiedfrom human or other mammalian source, or may be recombinantly orotherwise produced. Collagen of any type, including, but not limited to,types I, II, III, IV, or any combination thereof, may be used, althoughtype I is generally preferred. Either atelopeptide ortelopeptide-containing collagen may be used; however, when collagen froma xenogeneic source, such as bovine collagen, is used, atelopeptidecollagen is generally preferred, because of its reduced immunogenicitycompared to telopeptide-containing collagen. The collagen should be in apharmaceutically pure form such that it can be incorporated into a humanbody without generating any significant immune response.

Collagens for use in the present invention may be in the fibrillar ornonfibrillar form. Fibrillar collagens are generally preferred fortissue augmentation applications due to their increased persistence invivo. Nonfibrillar collagens, including chemically modified collagenssuch as succinylated or methylated collagen, may be preferable incertain situations, such as ophthalmic applications where an opticallytransparent material is required. Succinylated and methylated collagenscan be prepared according to the methods described in U.S. Pat. No.4,164,559 (which is hereby incorporated by reference in its entirety).Noncrosslinked collagens for use in the present invention are normallyin aqueous suspension at a concentration between about 20 mg/ml to about120 mg/ml, preferably, between about 30 mg/ml to about 80 mg/ml.Fibrillar collagen in suspension at various collagen concentrations iscommercially available from Collagen Corporation under the trademarkZyderm® I Collagen (35 mg/ml) and Zyderm II Collagen (65 mg/ml).

Collagen in its native state contains lysine residues having primaryamino groups capable of covalently binding with the hydrophobic andhydrophilic crosslinking agents of the invention and therefore need notbe chemically modified in any way prior to reaction with the desiredcrosslinking agent according to the methods of the invention.

Although intact collagen is preferred, denatured collagen, commonlyknown as gelatin, can also be used in the preparation of thecompositions of the invention.

Glycosaminoglycans for use in the present invention include, withoutlimitation, hyaluronic acid, chondroitin sulfate A, chondroitin sulfateC, dermatan sulfate, keratan sulfate, keratosulfate, chitin, chitosan,heparin, and derivatives or mixtures thereof. Glycosaminoglycans mustgenerally be modified, such as by deacetylation or desulfation, in orderto provide primary amino groups capable of binding with functionalgroups on hydrophobic and hydrophilic crosslinking agent according tothe methods of the present invention. Methods for chemically modifyingglycosaminoglycans by deacetylation and/or desulfation are described incommonly owned, copending U.S. application Ser. No. 08/146,843, filedNov. 3, 1993. In general, glycosaminoglycans can be deacetylated,desulfated, or both, as applicable, by the addition of a strong base,such as sodium hydroxide, to the glycosaminoglycan. Deacetylation and/ordesulfation provides primary amino groups on the glycosaminoglycan whichare capable of covalently binding with hydrophobic or hydrophiliccrosslinking agents according to the methods of the present invention.

Mixtures of various species of glycosaminoglycan, various types ofcollagen, or mixtures of various glycosaminoglycans with collagen may beused to prepare the crosslinked biomaterial compositions of the presentinvention.

If the final composition is intended for incorporation into the body ofa human or animal subject, biomaterials for use in the present inventionmust be in pharmaceutically pure form, or capable of being purified tobe in pharmaceutically pure form.

Preparation of Hydrophobic Crosslinking Agents

In order to prepare the crosslinked biomaterial compositions of thepresent invention, it is first necessary to provide a hydrophobicpolymer which contains, or can be derivatized to contain, two or moresuccinimidyl groups. As used herein, the term “hydrophobic polymer”refers to polymers which contain a relatively small proportion of oxygenor nitrogen atoms. As used herein, the term “containing two or moresuccinimidyl groups” is meant to encompass hydrophobic polymers whichare commercially available containing two or more succinimidyl groups,as well as those that must be chemically derivatized to contain two ormore succinimidyl groups. As used herein, the term “succinimidyl group”is intended to encompass sulfosuccinimidyl groups and other suchvariations on the “generic” succinimidyl group. The presence of thesodium sulfate moiety on the sulfosuccinimidyl group serves to increasethe solubility of the polymer.

Hydrophobic polymers for use in the present invention preferablycontain, or can be derivatized to contain, two or more succinimidylgroups, most preferably, two, three, or four succinimidyl groups. Thesesuccinimidyl groups are highly reactive with biomaterials containingprimary amino (—NH₂) groups, such as collagen and variousglycosaminoglycans and glycosaminoglycan derivatives.

Hydrophobic polymers which already contain two or more reactivesuccinimidyl groups include, without limitation, disuccinimidyl suberate(DSS), bis(sulfosuccinimidyl) suberate (BS³),dithiobis(succinimidylpropionate) (DSP),bis(2-succinimidooxycarbonyloxy)ethyl sulfone (BSOCOES), and3,3′-dithiobis(sulfosuccinimidylpropionate) (DTSPP), and their analogsand derivatives. The above-referenced polymers are commerciallyavailable from Pierce (Rockford, Ill.)+under catalog Nos. 21555, 21579,22585, 21554, and 21577, respectively. Structural formulas for theabove-referenced polymers, as well as generalized reaction productsobtained by reacting each of these polymers with collagen, are shown inFIGS. 1-5, respectively.

Certain polymers, such as polyacids, can be derivatized to contain twoor more reactive succinimidyl groups. Polyacids for use in the presentinvention include, without limitation, trimethylolpropane-basedtricarboxylic acid, di(trmethylol propane)-based tetracarboxylic acid,heptanedioic acid, octanedioic acid (suberic acid), and hexadecanedioicacid (thapsic acid). Many of these polyacids are commercially availablefrom DuPont Chemical Company.

According to a general method, polyacids can be chemically derivatizedto contain two or more succinimidyl groups by reaction with anappropriate molar amount of N-hydroxysuccinimide (NHS) in the presenceof N,N′-dicyclohexylcarbodiimide (DCC).

Polyalcohols such as trimethylolpropane and di(trimethylol propane) canbe converted to carboxylic acid form using various methods, then furtherderivatized by the addition of succinimidyl groups, as shown in FIGS. 6and 7.

Trimethylolpropane can be derivatized to tricarboxylic acid form, thenfurther derivatized by reaction with NHS in the presence of DCC toproduce a trifunctional crosslinking agent (ie., a compound having threesuccinimidyl groups available for reaction with various biomaterials),as shown in FIG. 6.

Di(trimethylol propane) can be derivatized to tetracarboxylic acid form,then further derivatized by reaction with NHS in the presence of DCC toproduce a tetrafunctional crosslinking agent, as shown in FIG. 7.

Other polyacids can be chemically derivatized to contain two or morereactive succinimidyl groups using methods similar to those shown inFIGS. 6 and 7 for trimethylolpropane-based tricarboxylic acid anddi(trimethylolpropane)-based tetracarboxylic acid, respectively.Polyacids such as heptanedioic acid (HOOC—(CH₂)₅—COOH), octanedioic acid(HOOC—(CH₂)₆—COOH), and hexadecanedioic acid (HOOC—CH₂)₁₄—COOH) arederivatized by the addition of succinimidyl groups to producedifunctional crosslinking agents.

Polyamines such as ethylenediamine (H₂N—CH₂ CH₂—NH₂),tetramethylenediamine (H₂N—CH₂)₄—NH₂), pentamethylenediamine(cadaverine) (H₂N—(CH₂)₅—NH₂), hexamethylenediamine (H₂N—(CH₂)₆—NH₂),bis(2-hydroxyethyl)amine (HN-(CH₂CH₂0H)₂), bis(2)aminoethyl)amine(HN—(CH₂CH₂NH₂)₂), and tris(2-aminoethyl)amine (N—(CH₂CH₂NH₂)₃) can bechemically derivatized to polyacids, which can then be derivatized tocontain two or more succinimidyl groups by reacting with the appropriatemolar amounts of N-hydroxysuccinimide in the presence of DCC accordingto the general method described above for polyacids. Many of thesepolyamines are commercially available from DuPont Chemical Company.

Preferred hydrophobic polymers for use in the invention, whether theyare commercially available containing two or more succinimidyl groups ormust be chemically derivatized to contain two or more succinimidylgroups, generally have a carbon chain that is no longer than about 14carbons. Polymers having carbon chains substantially longer than 14carbons generally have very poor solubility in aqueous solutions and, assuch, have very long reaction times when mixed with an aqueous solutionof a biomaterial such as collagen.

Preparation of Crosslinked Biomaterial Compositions Using HydrophobicCrosslinking Agents

In a general method for preparing the crosslinked biomaterialcompositions of the invention, a biomaterial which contains, or has beenchemically derivatized to contain, primary amino groups is mixed with ahydrophobic polymer which contains, or has been derivatized to contain,two or more succinimidyl groups capable of crosslinking the biomaterialby reacting with nucleophilic primary amino groups on the biomaterial.The hydrophobic crosslinking agent can be stored and used in either dryform or in solution, but is preferably used in dry form. Thecrosslinking agent may be mixed with either an aqueous solvent or ahydrophobic solvent prior to mixing with the biomaterial. If an aqueoussolvent is used, the crosslinking agent should be mixed with the solventjust prior to use, as the succinimidyl groups are reactive withneutrophiles such as oxygen and water. Exposure to aqueous solvents forextended periods of time will result in loss of crosslinking ability dueto hydrolysis of the crosslinking agent.

The biomaterial and hydrophobic crosslinking agent (in dry form) may bestored in separate syringes and then mixed using syringe-to-syringemixing techniques, as follows: the biomaterial and crosslinking agentare mixed by connecting the syringe containing the biomaterial with thesyringe containing the crosslinking agent using a syringe connector(such as a three-way stopcock) and passing the material back and forthbetween the two syringes until the material is adequately mixed (usuallyrequiring a minimum of about 20 passes, with one pass being counted eachtime the volume of material passes through the syringe connector).During the mixing process, crosslinking is initiated between moleculesof the biomaterial and the crosslinking agent.

The concentration of the hydrophobic crosslinking agent used in thepractice of the invention will vary depending upon a number of factors,including the type and molecular weight of the crosslinking agent used,the type and concentration of biomaterial used, and the degree ofcrosslinking desired. In general, we have found that hydrophobiccrosslinking agent concentrations in the range of about 0.1 to about 2percent by weight of the final composition are preferred to prepare thecrosslinked biomaterial compositions of the present invention.

Preparation of Heterogeneous Crosslinked Biomaterial Compositions UsingMixtures of Hydrophobic and Hydrophilic Crosslinking Agents

In a general method for preparing the heterogeneous crosslinkedbiomaterial compositions of the invention, a biomaterial which contains,or has been chemically derivatized to contain, primary amino groups iscombined and allowed to covalently bond with a mixture of hydrophobicand hydrophilic crosslinking agents. Preferably, the mixture ofhydrophobic and hydrophilic crosslinking agents is stored and used indry form, to prevent loss of crosslinking activity due to hydrolysis.The hydrophobic and hydrophilic crosslinking agents will generally notreact with one other because both crosslinking agents contain the samereactive groups (i e., succinimidyl groups) which preferentially bind toprimary amino groups on various biomaterials such as collagen andderivatized glycosaminoglycans.

In an alternative method, the biomaterial is mixed first with either thehydrophobic or hydrophilic crosslinking agent, then (preferably in rapidsuccession, before gellation occurs), with the other type ofcrosslinking agent.

As used herein, the term “hydrophobic polymer” refers to polymers whichcontain a relatively small proportion of oxygen or nitrogen atoms.Hydrophobic polymers which contain, or have been derivatized to contain,two or more reactive succinimidyl groups are the preferred hydrophobiccrosslinking agents for use in the preparation of the heterogeneouscrosslinked biomaterial compositions of the invention.

As used herein, the term “hydrophilic polymer” refers to polymers whichcontain a relatively large proportion of oxygen and/or nitrogen atoms,which serve to attract water molecules for hydrogen bonding. Synthetichydrophilic polymers, such as functionally activated polyethyleneglycols, are the preferred hydrophilic crosslinking agents for use inthe preparation of the heterogeneous crosslinked biomaterialcompositions of the present invention. Various activated forms ofpolyethylene glycol are described in detail in commonly owned U.S. Pat.No. 5,328,955 (the disclosure of which is incorporated herein byreference) and copending U.S. application Ser. No. 08/344,040, filedNov. 23, 1994.

Synthetic hydrophilic polymers for use in the present invention arepreferably multifunctionally activated and, more preferably,difunctionally activated. Preferred synthetic hydrophilic polymers aredifunctionally activated forms of PEG succinimidyl glutarate (SG-PEG),PEG succinimidyl (SE-PEG; referred to only as “S-PEG” in the '955patent), PEG succinimidyl succinamide (SSA-PEG), and PEG succinimidylcarbonate (SC-PEG). Reaction of SG-PEG with a biomaterial such ascollagen results in covalently bound conjugates containing an esterlinkage; reaction of SE-PEG (n=1−3) or SC-PEG (n=0) with a biomaterialresults in conjugates containing an ether linkage; and reaction ofSSA-PEG (n=1−10) with a biomaterial results in conjugates containing anamide linkage. The amide and ether linkages are generally lesssusceptible to hydrolysis than the ester linkage, and therefore mayresult in crosslinked biomaterial compositions having greater stabilityand persistence in vivo, depending on the environment into which theimplant material is placed. Ether linkages are susceptible to oxidation,and may be sensitive to degradation by free radicals.

Many of the activated forms of poly ethylene glycol described above arenow available commercially from Shearwater Polymers, Huntsville, Ala.,and from Union Carbide, South Charleston, West Va.

Use and Administration

The crosslinked biomaterial compositions of the present invention areparticularly useful in the preparation of formed implants for use in avariety of medical applications, including various artificial organs andtubular implants for use as vascular grafts and/or stents. In a generalmethod for preparing a formed implant, a biomaterial/crosslinking agentreaction mixture, prepared as described above, is extruded into molds ofvarious sizes and shapes, preferably before significant crosslinking hasoccurred between the biomaterial and the crosslinking agent (or mixtureof crosslinking agents). This period of time will vary depending uponthe type and concentration of both the biomaterial and the crosslinkingagent(s) used, but is generally within the range of about 5 to about 60minutes. The material should be removed from the mold only afteradequate time has elapsed to allow for equilibrium crosslinking to occurbetween the biomaterial and crosslinking agent(s). If necessary,residual, unbound crosslinking agent can be removed from the implantprior to its incorporation into the body of a patient.

The biomaterial/crosslinking agent mixture can also be applied to (forexample, by extrusion, dipping, brushing, or painting) onto one or moresurface of a preformed synthetic implant, such as a bone prosthesis orsynthetic vascular graft or stent, and allowed to crosslink in place,thereby providing a crosslinked, nonimmunogenic biomaterial coating onthe surface of the implant. Alternatively, all or part of a preformedsynthetic implant can be dipped into a container holding thebiomaterial/crosslinking agent reaction solution.

The biomaterial/crosslinking agent mixture can be extruded in the shapeof a string and allowed to crosslink in that configuration. When thestrings are fully crosslinked, they can be dried to remove substantiallyall unbound water. The dried strings can be inserted through a needle toa dermal site in need of correction (such as a depressed scar orwrinkle) in order to provide soft tissue augmentation. The dried stringscan also be chopped into fine pieces, suspended in a nonaqueous carrier,and injected to a tissue site in need of augmentation, which may be adermal site or other soft tissue site such as an inadequatelyfunctioning sphincter (e.g., urinary, anal, or esophageal sphincter).When exposed to biological fluids, the crosslinked strings willrehydrate in situ and swell to approximately five times their drieddiameter. The dried strings can also be used as suture materials, orbraided, knit, or woven to provide biomaterials for tendon or ligamentrepair or replacement.

A suitable particulate material, such as ceramic particles, can be mixedwith the biomaterial prior to mixing with the crosslinking agent toprovide a material suitable for hard tissue augmentation, such as therepair or replacement of bone or cartilage. These materials can beadministered in fluid form (prior to crosslinking) to the site of a boneor cartilage defect and allowed to crosslink in place, or can be used toprepare formed bone or cartilage implants (using techniques similar tothose described above for the preparation of formed implants for softtissue repair) which can then be molded or cut to the desired size andshape.

The crosslinked biomaterial compositions of the invention can also beused as injectable formulations in the augmentation of soft or hardtissues of the body. Following mixing of the biomaterial and thecrosslinking agent(s), the reaction mixture should be injected to atissue site before significant crosslinking has occurred, to preventblockage of the syringe needle with the crosslinked composition. If thematerial is injected to a tissue site before equilibrium crosslinkinghas occurred, functional groups on the crosslinking agent(s) may bind tocollagen molecules in the host tissue, thereby providing biologicalanchoring of the biomaterial to the host tissue. Implants which havebeen “biologically anchored” to host tissue are more difficult todisplace and therefore may show greater persistence in vivo thancurrently available injectable biomaterial compositions.

Biologically active agents, such as cytokines or growth factors, can beincorporated into the compositions of the invention, either by simpleadmixture, or by covalently binding the active agent to the crosslinkingagent prior to combining the crosslinking agent with the biomaterial.The active agents may serve to recruit cells to the area of the implant,further anchoring the implant to host tissue, and may accelerate woundhealing when adminstered to a wound site.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the preferred embodiments of the conjugates, compositions, anddevices and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, molecularweight, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1

Preparation and Characterization of Crosslinked Collagen CompositionsUsing Hydrophobic Crosslinking Agents

Fibrillar collagen (Zyderm® I Collagen, available from CollagenCorporation, Palo Alto, Calif.) and methylated (nonfibrillar) collagen(prepared by reacting fibrillar collagen with methanol for approximately1-3 days at 21° C.) were crosslinked using disuccinimidyl suberate(DSS), bis(sulfosuccinimidyl) suberate (BS³), difunctionally activatedSE-PEG (n=2, 3800 MW), and difunctionally activated SG-PEG (3800 MW).

The fibrillar collagen formulations were prepared by mixing the contentsof a 1-cc syringe containing 1.0 cc of Zyderm Collagen (35 mg/mlcollagen concentration) with the contents of a 1-cc syringe containingone of the following crosslinking agents in the quantity specified:

-   -   3 mg of DSS;    -   3 mg of BS³;    -   5 mg of SE-PEG; or    -   5 mg of SG-PEG.

The methylated collagen formulations were prepared by mixing thecontents of a 1-cc syringe containing 1.0 cc of methylated collagen (21mg/ml collagen concentration) with the contents of a 1-cc syringecontaining one of the following crosslinking agents in the quantityspecified:

-   -   3 mg of DSS;    -   3 mg of BS³;    -   10 mg of SE-PEG; or    -   10 mg of SG-PEG.

All of the crosslinking agents were used in dry form. The collagen andcrosslinking agent were mixed by passing the, material between the twosyringes using a 3-way stopcock, employing about 40 to 50 passes ofmaterial between the syringes. Once adequate mixing of the collagen andcrosslinking agent had been achieved, the material was transferred intoone syringe and incubated at 37° C. for approximately. 16 hours.

Each of the crosslinked collagen materials prepared as described abovewas extruded out of the plunger end of its syringe. The resultingcrosslinked cylindrical gels were then sectioned into 5-mm thick disks.Each of the formulations was then evaluated according to some or all ofthe following test methods: differential scanning calorimetry (DSC),solubilization in 1 mg/ml trypsin solution, and oxidative degradation in3% hydrogen peroxide (H₂O₂). The results of these evaluations arepresented in Table 1, below.

TABLE 1 Characterization of Various Crosslinked Collagen CompositionsSolubilization in Oxidative Degradation Material DSC (Tm ° C.) TrypsinSolution in 3% H₂O₂ DSS-ZI 74.3 7 days 14 days DSS-MC 57.7 2 days N/ABS³-ZI 67.6 N/A N/A BS³-MC 58.6/64 N/A N/A SEPEG-ZI N/A 3 days 10 daysSEPEG-MC N/A 16 hours N/A SGPEG-ZI 60.8 3 days   7 days SGPEG-MC N/A 16hours N/A ZI = Zyderm ® I Collagen (35 mg/ml collagen concentration) MC= methylated collagen (21 mg/ml collagen concentration) DSS =disuccinimidyl suberate BS³ = bis(sulfosuccinimidyl) suberate SEPEG =difunctionally activated SE-PEG (n = 2, 3800 MW) SGPEG = difunctionallyactivated SG-PEG (3800 MW) N/A = Data not available.

Differential scanning calorimetry (DSC) is used to measuredenaturational transitions in collagen, which can be used to assess therelative strength of crosslinking achieved. As indicated by the DSCresults above, crosslinking of fibrillar collagen by the hydrophobiccrosslinking agents DSS and BS³ is at least as strong as that achievedusing the hydrophilic crosslinking agent SG-PEG. Slightly lower numberswere obtained for the methylated (nonfibrillar) collagen formulations.

Solubilization in trypsin solution was determined by incubating a 5-mmthick disk of each crosslinked material at 37° C. in a solutioncomprising 1 mg trypsin in 1 ml water and measuring how much time wasrequired to disperse the crosslinked collagen gel. As shown above,approximately twice as much time (7 days) was required to solubilize theDSS-ZI gel as was required to solubilize the SEPEG-ZI and SGPEG-ZI gels(3 days each), indicating that DSS achieves stronger crosslinking (i.e.,increased crosslinking density) to fibrillar collagen than do eitherSE-PEG or SG-PEG. The methylated collagen formulations demonstrated lessstability in trypsin solution in general, but the methylated collagenformulations crosslinked using DSS showed considerable improvement instability over those crosslinked using either SE-PEG or SG-PEG.

Oxidative degradation was determined by incubating a 5-mm thick disk ofeach crosslinked material at 37° C. in a 3% solution of hydrogenperoxide in water and measuring how much time was required to dispersethe crosslinked collagen gel. As with the results of the trypsinsolubilization described above, nearly twice as much time (14 days) wasrequired to solubilize the DSS-ZI gel as was required to solubilize theSEPEG-ZI (10 days) and SGPEG-ZI gels (7 days), indicating that DSSachieves stronger crosslinking to fibrillar collagen than do eitherSE-PEG or SG-PEG. Thus, with regard to trypsin sensitivity andsusceptibility to oxidative degradation, the collagen materialscrosslinked using hydrophobic crosslinking agents showed considerableand unexpected improvement over those crosslinked with the hydrophiliccrosslinking agents previously described in the art.

Example 2

In vivo Persistence of Crosslinked Collagen Compositions

Crosslinked collagen formulations were prepared fresh by mixing thecontents of a 1-cc syringe containing 1.0 gram of a mixture of Zyplast®(glutaraldehyde-crosslinked collagen having a collagen concentration of35 mg/ml, available from Collagen Corporation, Palo Alto, Calif.) andZyderm® Collagens (in a 70:30 weight/ weight ratio) with the contents ofa 1-cc syringe containing either 3 mg of DSS, 3 mg of SE-PEG, or 3 mg ofSG-PEG. A noncrosslinked mixture of Zyplast and Zyderm Collagens in a70:30 weight ratio was used as the control. Two groups consisting of 24male Sprague-Dawley rats each were injected with implants consisting of0.5 milliliters each of two of the four formulations, according to theschedule below.

Animal Group A:

-   -   Site 1 Zyplast/Zyderm Collagen mixture (control)    -   Site 2 Zyplast/Zyderm Collagen mixture crosslinked using DSS

Animal Group B

-   -   Site 1 Zyplast/Zyderm Collagen mixture crosslinked using SG-PEG    -   Site 2 Zyplast/Zyderm Collagen mixture crosslinked using SE-PEG

The materials were injected subcutaneously through a 27-gauge needlewithin approximately 5 minutes of mixing the collagen and crosslinkingagent.

Six animals from each of Groups A and B were sacrificed at each of the7, 14, 28, and 90 day post-implantation time points. The implants withsurrounding tissue were excised and examined histologically. Theinjected crosslinked materials had assumed a discrete, football-shaped,bolus-like configuration, whereas the noncrosslinked formulation waspresent as a more diffuse mass. The implants from four animals out ofeach group were used for histology studies and wet weight experiments.The implants from two animals out of each group were used to measure themechanical force required to dislodge the implant from the host tissue.The results of the histology studies and wet weight experiments arediscussed below.

The excised implants were examined histologically and scored on a scaleof 0 through 4 on each of three parameters: inflammatory infiltrate,fibroblast ingrowth, and fibrosis. A score of 4 indicated the presenceof a maximum amount of a parameter; a score of 0 indicated that theparticular parameter was not observed in connection with the implantbeing examined (i.e., a score of 0 on inflammatory infiltrate indicatesthat no inflammatory infiltrate was observed in the implant site).Results of the histological examinations are presented in Tables 2, 3,and 4, and discussed below. Average scores are listed in parentheses.

TABLE 2 Inflammatory Infiltrate Implant Material Day 7 Day 14 Day 28 Day90 Z/Z 0, 2, 2, 1 2, 0, 0 0, 0, 0, 1 0, 0, 0, 0 (1.25) (0.67) (0.25) (0)Z/Z + DSS 1, 2, 2, 3, 2 3, 3, 1 1, 1, 1, 1 0, 0, 0, 0 (2,0) (2.3) (1.0)(0) Z/Z + SG-PEG 1, 1, 1 0, 1, 3, 1 1, 0, 2, 1 0, 0, 0, 0 (1.0) (1.25)(1.0) (0) Z/Z + SE-PEG 1, 1, 1, 2 0, 1, 1, 1 1, 0, 2, 2 0, 0, 0, 0(1.25) (0.75) (1.25) (0) Z/Z = mixture of Zyplast ® and Zyderm ® ICollagens in a 70:30 weight/weight ratio DSS = disuccinimidyl suberateBS³ = bis(sulfosuccinimidyl) suberate SEPEG = difunctionally activatedSE-PEG (n = 2, 3800 MW) SGPEG = difunctionally activated SG-PEG (3800MW)

At days 7 and 14, the collagen implants crosslinked using DSS showed amoderate inflammatory response, slightly greater than the responsesobserved for the other (crosslinked and noncrosslinked) collagencompositions. By day 28, inflammatory infiltrate into theDSS-crosslinked implant was minimal, diminishing to nonexistent by day90.

TABLE 3 Fibroblast Ingrowth Implant Material Day 7 Day 14 Day 28 Day 90Z/Z 0, 0, 0, 0 1, 1, 1 1, 0, 0, 1 0, 0, 1, 1 (0) (1.0) (0.5) (0,5) Z/Z +DSS 0, 0, 0, 0, 0 0, 0, 0 0, 0, 0, 0 0, 0, 0, 0 (0) (0) (0) (0) Z/Z +SG-PEG 0, 0, 0 1, 1, 1, 1 1, 0, 2, 1 0, 1, 1, 1 (0) (1.0) (1.0) (0.75)Z/Z + SE-PEG 0, 0, 0, 0 1, 1, 1, 0 1, 0, 2, 2 0, 0, 0, 1 (0) (0.75)(1.25) (0.25)

Unlike the other crosslinked and noncrosslinked collagen formulations,the DSS-crosslinked implants showed no evidence of fibroblast ingrowththroughout the entire duration of the study. This is most likely due tothe very tight crosslinked collagen network achieved using DSS as acrosslinking agent.

TABLE 4 Fibrosis Implant Material Day 7 Day 14 Day 28 Day 90 Z/Z 0, 1,1, 2 1, 0, 0 0, 0, 1, 0 0, 0, 0, 0 (1.0) (0.33) (0.25) (0) Z/Z + DSS 1,0, 2, 1, 2 1, 0, 1 0, 0, 1, 1 0, 0, 0, 0 (1.2) (0.67) (0.5) (0) Z/Z +SG-PEG 0, 1, 1 1, 1, 1, 1 1, 1, 1, 1 0, 0, 0, 0 (0.67) (1.0) (1.0) (0)Z/Z + SE-PEG 1, 0, 2, 1 0, 1, 1, 1 0, 1, 1, 1 0, 0, 0, 0 (1.25) (0.75)(0.75) (0)

Fibrosis was observed to be similar in all three of the crosslinkedcollagen compositions examined.

Each of the implants was weighed following explantation. Wet weight ofthe implant as a percentage of the original weight of the implant isshown in FIG. 8 for each of the four formulations at each time point.There were no significant differences between any of the formulations atthe 7, 14, and 28 day time points. However, at the 90-day time point,the collagen formulation crosslinked using DSS showed significantlybetter retention of wet weight (close to 100 percent) than the otherformulations. Due to the lack of fibroblast ingrowth seen duringhistological examination, the wet weight of the DSS-crosslinked implantis believed to consist substantially of the implant material itselfrather than invading cells. This observation indicates that theDSS-crosslinked collagen implants were not resorbed into the host tissueas quickly as the other collagen implant materials, possibly due to thetightly crosslinked network achieved using DSS as a crosslinking agent.

At each of the 7, 28, and 90-day time points of the study, the portionof the skin containing the implant was excised from two animals fromeach of Groups A and B. The skin surrounding the implant was trimmedinto a uniform rectangular shape having dimensions of 2 cm×4 cm. Theencapsulated tissue that had grown over the surface of the implant wasremoved so that the implant now appeared to be merely resting on thesurface of the dermis.

The piece of skin containing the implant was pinned to a 3 cm×5 cmwooden board using one thumbtack at each of the four corners of theskin. As illustrated in FIG. 9, a sling was placed externally around theperimeter of the implant. The mechanical force required to dislodge theimplant from the tissue was measured using the Instron Universal Tester,Model 4202, by holding the wooden board (to which the piece of skin wasattached) in one of the Instron's clamps and holding the end of thesling in the other clamp. The Instron pulled on the clamp holding thesling until the implant broke free from the tissue. Force anchoring totissue is depicted graphically in FIG. 10 for each of the fourformulations at the 7, 28, and 90-day time points. There were nosignificant differences between the formulations crosslinked using thehydrophobic crosslinking agent (DSS) and the formulations crosslinkingusing either of the hydrophilic crosslinking agents (SE-PEG; SG-PEG).

Example 3

Preparation and Characterization of Crosslinked Biomaterial CompositionsContaining Mixtures of Hydrophobic and Hydrophilic Crosslinking Agents

Fibrillar collagen (Zyderm® I Collagen, 35 mg/ml collagen concentration,available from Collagen Corporation, Palo Alto, Calif.) was crosslinkedusing disuccinimidyl suberate (DSS), difunctionally activated SE-PEG(n=2, 3800 MW), and a 50:50 (weight/weight) mixture of DSS anddifunctionally activated SE-PEG. The crosslinked collagen formulationswere prepared by mixing the contents of a 5-cc syringe containing 5.0grams of Zyderm Collagen with the contents of a 5-cc syringe containingeither 15 mg of DSS, 15 mg of SE-PEG, or 15 mg of the DSS/SE-PEGmixture.

All of the crosslinking agents were used in dry form. The DSS/SE-PEGmixture was prepared immediately prior to crosslinking by placing 7.5 mgeach of DSS and SE-PEG into a 5-cc syringe, then shaking the syringe byhand to mix the two crosslinking agents.

The collagen and crosslinking agent were mixed by passing the materialbetween the two syringes using a 3-way stopcock, employing about 40 to50 passes of material between the syringes. Once adequate mixing of thecollagen and crosslinking agent had been achieved, the material wastransferred into one syringe and incubated at 37° C. for approximately16 hours.

Each of the three crosslinked collagen materials prepared as describedabove was extruded out of the plunger end of its syringe. The resultingcrosslinked cylindrical gels were then sectioned into 5-mm thick disks.The three formulations were evaluated using differential scanningcalorimetry (DSC). The gel strength of each formulation was measuredusing the Instron Universal Tester, Model 4202. DSC and gel strengthresults for each of the three crosslinked collagen formulations arepresented in Table 5, below.

TABLE 5 DSC and Gel Strength Results for Various Crosslinked CollagenCompositions Average Crosslinking Gel Strength Gel Strength Agent DSC (°C.) (Newtons) (Newtons) S.D. DSS 74.3 54.1 46.5 6.0 40.5 45.0 41.5 51.1SE-PEG 59.5 59.4 58.6 2.5 56.4 56.2 58.7 62.4 DSS/SE-PEG 53-65*  28.141.7 8.2 65-80** 43.9 44.2 50.3 42.3 *Broad main peak. **Broad shoulderpeak.

The inconsistency in the DSC and gel strength results for the collagencomposition prepared using a mixture of hydrophobic and hydrophiliccrosslinking agents may be due to several factors, among them:insufficient mixing of the two crosslinking agents prior to mixing withcollagen, the heterogeneous nature of the composition itself, and,possibly, a ratio of crosslinking agents that had not been optimized.Another factor may be that the SE-PEG is able to crosslink collagen morequickly-than DSS due to the lower solubility of the DSS in the aqueoussolution in which the collagen fibers are suspended.

Collagen compositions prepared using mixtures of hydrophobic andhydrophilic crosslinking agents may be useful in certain therapeuticapplications due to the relative contributions of the two differenttypes of crosslinking agent to the properties of the final composition:the hydrophobic crosslinking agent, increased stability; the hydrophiliccrosslinking agent, increased elasticity and better overall handlingproperties.

It is not intended that the invention be limited by the preferredembodiments described above, which are used for purposes ofillustration. The invention is intended to have the scope defined by theattached claims.

1. An injectable, crosslinkable composition that crosslinks in situfollowing administration to a patient to form a heterogeneous,crosslinked biomaterial composition, comprising: a biomaterial comprisedof a biocompatible polymer containing nucleophilic groups; a hydrophobiccrosslinking agent containing up to about 14 carbon atoms and comprisedof a polyacid esterified with a reactive moiety selected from the groupconsisting of succinimidyl and sulfosuccinimidyl groups; and ahydrophilic crosslinking agent, wherein both the hydrophobiccrosslinking agent and the hydrophilic crosslinking agent crosslink thebiomaterial in situ following administration of the composition to apatient, but are not reactive with respect to each other.
 2. Thecomposition of claim 1, further including water, such that thecomposition is comprised of an aqueous suspension.
 3. The composition ofclaim 1, wherein the nucleophilic groups are primary amino groups. 4.The composition of claim 1, wherein the biomaterial is selected from thegroup consisting of collagen, glycosaminoglycans, and mixtures thereof.5. The composition of claim 4, wherein the biomaterial is comprised ofcollagen.
 6. The composition of claim 5, wherein the collagen isfibrillar collagen.
 7. The composition of claim 5, wherein the collagenis nonfibrillar collagen.
 8. The composition of claim 7, wherein thenon-fibrillar collagen is comprised of chemically derivatized collagenselected from the group consisting of succinylated collagen andmethylated collagen.
 9. The composition of claim 8, wherein the collagenis succinylated collagen.
 10. The composition of claim 8, wherein thecollagen is methylated collagen.
 11. The composition of claim 5, whereinthe collagen is selected from the group consisting of Type I collagen,Type II collagen, Type III collagen, Type IV collagen, and mixturesthereof.
 12. The composition of claim 11, wherein the collagen is Type Icollagen.
 13. The composition of claim 5, wherein the collagen istelopeptide collagen.
 14. The composition of claim 5, wherein thecollagen is atelopeptide collagen.
 15. The composition of claim 5,wherein the collagen is denatured collagen.
 16. The composition of claim4, wherein the biomaterial is a glycosaminoglycan.
 17. The compositionof claim 16, wherein the glycosaminoglycan is selected from the groupconsisting of hyaluronic acid, chondroitin sulfate A, chondroitinsulfate C, dermatan sulfate, keratan sulfate, keratosulfate, chitin,chitosan, heparin, and derivatives thereof.
 18. The composition of claim1, wherein the hydrophobic crosslinking agent is disuccinimidylsuberate.
 19. The composition of claim 1, wherein the hydrophobiccrosslinking agent is comprised of a polyacid containing two, three orfour carboxylic acid groups each esterified with a succinimidyl group.20. The composition of claim 19, wherein the polyacid is selected fromthe group consisting of trimethylolpropane tricarboxylic acid,di(trimethylol propane) tetracarboxylic acid, heptanedioic acid, subericacid and hexadecanedioic acid.
 21. The composition of claim 1, whereinthe hydrophobic crosslinking agent is comprised of a polyacid containingtwo, three or four carboxylic acid groups each esterified with asulfosuccinimidyl group.
 22. The composition of claim 21, wherein thepolyacid is selected from the group consisting of trimethylolpropanetricarboxylic acid, di(trimethylol propane) tetracarboxylic acid,heptanedioic acid, suberic acid and hexadecanedioic acid.
 23. Thecomposition of claim 1, wherein the hydrophobic crosslinking agent isselected from the group consisting of disuccinimidyl suberate,dithiobis(succinimidyl-propionate, bis(sulfosuccinimidyl) suberate,bis(2-succinimidooxycarbonyloxy)ethyl sulfone, and3,3′-dithiobis(sulfosuccinimidylpropionate).
 24. The composition ofclaim 1, wherein the hydrophilic crosslinking agent is comprised of anactivated hydrophilic polymer.
 25. The composition of claim 24, whereinthe activated hydrophilic polymer is selected from the group consistingof polyethylene glycol succinimidyl glutarate, polyethylene glycolsuccinimidyl, polyethylene glycol succinimidyl succinamide, andpolyethylene glycol succinimidyl carbonate.
 26. The composition of claim24, wherein the activated hydrophilic polymer is polyethylene glycolsuccinimidyl glutarate.
 27. The composition of claim 24, wherein theactivated hydrophilic polymer is polyethylene glycol succinimidyl.
 28. Akit for administering an in situ crosslinkable composition to a patientvia injection, comprising: a first syringe containing a biomaterialcomprised of a biocompatible polymer containing nucleophilic groups; asecond syringe containing a mixture of a hydrophilic crosslinking agentand a hydrophobic crosslinking agent containing up to about 14 carbonatoms and comprised of a polyacid esterified with reactive moietiesselected from the group consisting of succinimidyl groups andsulfosuccinimidyl groups, wherein both the hydrophobic crosslinkingagent and the hydrophilic crosslinking agent crosslink the biomaterialin situ following administration of a combination of the biomaterial andthe crosslinking agents to a patient, but are not reactive with respectto each other.
 29. The kit of claim 28, wherein the first syringefurther contains water, such that the biomaterial is in an aqueoussuspension.
 30. The kit of claim 28, wherein the nucleophilic groups areprimary amino groups.
 31. The kit of claim 28, wherein the biomaterialis selected from the group consisting of collagen, glycosaminoglycans,and mixtures thereof.
 32. The kit of claim 31, wherein the biomaterialis comprised of collagen.
 33. The kit of claim 32, wherein the collagenis fibrillar collagen.
 34. The kit of claim 32, wherein the collagen isnonfibrillar collagen.
 35. The kit of claim 34, wherein the nonfibrillarcollagen is comprised of chemically derivatized collagen selected fromthe group consisting of succinylated collagen and methylated collagen.36. The kit of claim 35, wherein the collagen is succinylated collagen.37. The kit of claim 35, wherein the collagen is methylated collagen.38. The kit of claim 32, wherein the collagen is selected from the groupconsisting of Type I collagen, Type II collagen, Type III collagen, TypeIV collagen, and mixtures thereof.
 39. The kit of claim 38, wherein thecollagen is Type I collagen.
 40. The kit of claim 32, wherein thecollagen is telopeptide collagen.
 41. The kit of claim 32, wherein thecollagen is atelopeptide collagen.
 42. The kit of claim 32, wherein thecollagen is denatured collagen.
 43. The kit of claim 31, wherein thebiomaterial is a glycosaminoglycan.
 44. The kit of claim 43, wherein theglycosaminoglycan is selected from the group consisting of hyaluronicacid, chondroitin sulfate A, chondroitin sulfate C, dermatan sulfate,keratan sulfate, keratosulfate, chitin, chitosan, heparin, andderivatives thereof.
 45. The kit of claim 28, wherein the hydrophobiccrosslinking agent is comprised of a polyacid containing two, three orfour carboxylic acid groups each esterified with a succinimidyl group.46. The kit of claim 28, wherein the hydrophobic crosslinking agent iscomprised of a polyacid containing two, three or four carboxylic acidgroups each esterified with a sulfosuccinimidyl group.
 47. The kit ofclaim 45, wherein the polyacid is selected from the group consisting oftrimethylolpropane tricarboxylic acid, di(trimethylol propane)tetracarboxylic acid, heptanedioic acid, suberic acid andhexadecanedioic acid.
 48. The kit of claim 46, wherein the polyacid isselected from the group consisting of trimethylolpropane tricarboxylicacid, di(trimethylolpropane) tetracarboxylic acid, heptanedioic acid,suberic acid and hexadecanedioic acid.
 49. The kit of claim 28, whereinthe hydrophobic crosslinking agent is selected from the group consistingof disuccinimidyl suberate, dithiobis(succinimidylpropionate,bis(sulfosuccinimidyl) suberate, bis(2-succinimidooxycarbonyloxy)ethylsulfone, and 3,3-dithiobis(sulfosuccinimidylpropionate).
 50. The kit ofclaim 49, wherein the hydrophobic crosslinking agent is disuccinimidylsuberate.
 51. The kit of claim 28, wherein the hydrophilic crosslinkingagent is comprised of an activated hydrophilic polymer.
 52. The kit ofclaim 51, wherein the activated hydrophilic polymer is selected from thegroup consisting of polyethylene glycol succinimidyl glutarate,polyethylene glycol succinimidyl, polyethylene glycol succinimidylsuccinamide, and polyethylene glycol succinimidyl carbonate.
 53. The kitof claim 52, wherein the activated hydrophilic polymer is polyethyleneglycol succinimidyl glutarate.
 54. The kit of claim 52, wherein theactivated hydrophilic polymer is polyethylene glycol succinimidyl.